Yr10 gene polymorphism in bread wheat varieties

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1 African Journal of Biotechnology Vol. 7 (14), pp , 18 July, 2008 Available online at ISSN Academic Journals Full Length Research Paper Yr10 gene polymorphism in bread wheat varieties A. Temel 1 *, F. entürk-akfırat 2, F. Erturul 3, A. Yumurtacı 3, Y. Aydın 4, T. Talas-Ora 3, N. Gözükırmızı 1*, N. Bolat 5, Ö. Yorgancılar 5, S. Belen 5, M. Yıldırım 5, M. Çakmak 5, E. Özdemir 5, L. Çetin 6, Z. Mert 6, H. Sipahi 6, S. Albustan 6, K. Akan 6, F. Düünceli 6 and A. Altınkut Uncuolu 3 1 Istanbul University, Molecular Biology and Genetics Department, 34118, Vezneciler, Istanbul/Turkey. 2 Gebze Institute of Technology, Institute of Higher Technologies, Biology Department, Muallimköy Campus, Gebze- Kocaeli/Turkey. 3 TUBITAK, MRC, Institute for Genetic Engineering and Biotechnology, P.O. Box , Gebze-Kocaeli/Turkey. 4 Marmara University, Biology Department, Göztepe 34722, Kadıköy, Istanbul/Turkey. 5 Anatolian Agricultural Research Institute PK 17, 26010, Eskiehir/Turkey. 6 Field Crops Research Institute, P.K. 226, Ulus, Ankara/Turkey. Accepted 20 June, 2008 Yellow rust resistance locus Yr10 located on chromosome 1B in Moro and originated from the Turkish line PI was investigated in terms of polymorphism in seven winter type bread wheat cvs. (Triticum aestivum ssp. Aestivum) Altay2000, zgi2001, Sönmez2001 (yellow rust resistant), Aytın98, ES14, Harmankaya99 (yellow rust susceptible) and PI as control. Exon 1 (1-833 bp) and Exon 2 ( bp) parts of Yr10 were amplified with three primers. Amplification was not observed with E2A primers in Harmankaya99, zgi2001 and Sönmez2001 cvs, while amplification products were observable at all tested varieties with the other primers. PCR results showed that E2A reverse primer is not able to anneal to the three varieties mentioned above. Sequence analysis and bioinformatics analysis proved that there has been single nucleotide changes especially in the second exon. The most similar sequences to the first exon of Harmankaya99, zgi01 and Sönmez2001 are AF (Aegilops tauschii NBS-LRR-like gene), AF (A. tauschii NBS-LRR-like gene sequence) and AF509534, respectively. These results could be helpful in revealing divergence between resistant and susceptible varieties. Key words: Triticum aestivum L., yellow rust, resistance gene, PCR, sequence analysis. INTRODUCTION Wheat (Triticum aestivum ssp. aestivum) is one of the most important cereal crops in the world for both human food and animal feed. Characterization of disease resistance genes has great importance for the transfer of agronomically important genes to commercial varieties. Gene based molecular markers (Peng et al., 2000; Wang et al., 2002; Chen et al., 2003) when using PCR based gene specific primers, present an opportunity not only for selection of desired varieties but also provide the information about sequence polymorphisms in genes. How- *Corresponding author. asl_temel@yahoo.com. Tel: / ever direct sequence information gives more valuable data for understanding of resistance mechanisms. Yellow rust or stripe rust caused by the fungus Puccinia striiformis f.sp. tritici, is one of the most damaging diseases affecting bread wheat in temperate regions (Mallard et al., 2005). Pathogen utilizes water and nutrients of the host and reduces leaf area and yield. Growing resistant varieties is the most effective and economically method of disease control (Röbbelen and Sharp, 1978; Line and Chen, 1995). In this research, we investigated sequence variations using bioinformatics tools in Yr10 locus which is one of the main resistance genes to yellow rust (stripe rust) caused by P. striiformis from seven winter type bread wheat cvs.

2 Temel et al Table 1. Primers used in the study. Primer name Forward (5 3 ) Reverse (5 3 ) Product (bp) E1 CTTGCTGGCGACCTGCTTA TGTTTCGCTCCACGCTGACT 754 E2 Upstream TGGTAGTAGAGTAATCGCAACA TCTTCAGATTTGGAGGTAGG 377 E2A Downstream TGGAAATGGATAGGCGAAGG AAATCAATGAAGCCGCAACC 872 MATERIALS AND METHODS Plant material Altay2000, zgi2001, Sönmez2001 (Yellow rust resistant), Aytın98, ES14, Harmankaya99 (Yellow rust sensitive) and PI (Control) were screened for Yr10 gene polymorphisms. Seeds were obtained from Anatolian Agricultural Research Institute. Structure of Yr10 Yr10 is found in Turkish line PI and is also the first yellow rust resistance gene, which is sequenced (GenBank No: AF149112) (Authors; Laroche A, Frick, M.M., Huel, R., Nykiforuk, C., Conner, B., Kuzyk, A.). Yr10 is a dominant gene which confers race specific resistance to yellow rust. It is 3630 bp long and consists of two exons interrupted by an intron. Its mrna transcript is 2475 bps long (GenBank No: AF149114). Primer design PCR primers were designed to amplify both exon 1 and exon 2 using the program Primer Premier Version One pair (E1) was designed for the first exon, two pairs (E2, E2A) were designed for the second exon (Table 1). PCR analysis Genomic DNA was extracted from leaves of 23 day-old seedlings according to Song and Henry (1995). Genomic DNA was quantified spectrophotometrically. PCR was performed at 25 µl final volume containing 1X enzyme buffer, 1.5 mm MgCl 2, 0.2 mm of each dntp, 2 µm forward and 2 µm reverse primer, 100 ng template DNA and 1 U Taq polymerase (M186A, Promega). Amplifications were performed with an initial denaturation at 94 o C for 2 min, 30 cycles of 94 o C for 30 s, 55 o C for 30 s (E1, E2A primers) or 50 o C for 30 s (E2 primers) or 60 o C (E2 forward and E2A reverse primers), 72 o C for 1 min and completed after a final elongation at 72 o C for 7 min. PCR products were run on 1% nusieve agarose gels and stained with 0.5 µg/ml ethidium bromide. Sequencing PCR products were excised from agarose gel and recovered using a purification kit (Wizard SV Gel and PCR Clean-Up System A9281, Promega). After the checking of purified bands on agarose gels, 754 bp products from 7 varieties and 1311 bp product from 4 varieties were sent to sequencing (IONTEK). Multiple sequence alignments were calculated using CLUSTALW (Thompson et al., 1994) ( First and second exon sequences were compared in the nucleotide collection database (nr/nt) using BLASTN (discontiguous megablast) (Altschul et al., 1990) ( RESULTS AND DISCUSSION PCR amplification E1 and E2 primer pairs gave the expected product size in 7 varieties (Figure 1a, b). But E2A primer did not amplify any product in 3 varieties (Harmankaya99, zgi2001, Sönmez2001) (Figure 1 c). In order to find out which primer could not bind to template, E2 forward primer (upstream region of the second exon) and E2A reverse primer (downstream region of the second exon) were used together. After gradient PCR, a band of expected size (1300 bp) was amplified at 60 o C annealing temperature in 4 varieties (PI178383, Altay2000, Aytın98, ES14) (Figure 1d). We hypothesized E2A reverse primer cannot bind to genomic DNA in 3 varieties mentioned above. Multiple alignment The most similar varieties to the first exon of Yr10 are PI (98%) and Altay2000 (99%) (Table 2). Harmankaya99, zgi01 and Sönmez2001 are the least similar varieties to the first exon of Yr10 (Table 2). The most similar variety to the second exon of Yr10 is PI (97%) (Table 3). Multiple alignment results are also presented as phylogram trees (Figures 2 and 3). The best BLASTN matches for exon 1 sequence of Harmankaya99, zgi01 and Sönmez2001 are AF (Aegilops tauschii NBS-LRR-like gene) (91%), AF (Aegilops tauschii NBS-LRR-like gene sequence) (95%) and AF (98%) respectively. Spielmeyer and Lagudah (2003) hybridized probe RgaYr10 to genomic DNA of A. tauschii line AUS They isolated four clones representing at least four different RgaYr10 gene family members. These clones were sequenced and submitted to GenBank as AF509533, AF and AF The results obtained from this work indicate that (1) Yr10 gene sequence is present in all of these varieties, (2) the divergence between the varieties is raised from the variations in the second exon and (3) the first exon is

3 2330 Afr. J. Biotechnol. Μ C Μ C a b Μ C Μ C c d Figure 1. Amplification products of E1 (a), E2 (b), E2A (c) and E2 forward and E2A reverse (d) primers. Product length 754 bp, 377 bp, 872 bp and 1311 bp respectively. M. Marker, 1. PI178383, 2. Altay2000, 3. Aytın98, 4. ES14, 5. Harmankaya99, 6. zgi2001, 7. Sönmez2001, C - Negative control. Table 2. Comparison of the first exon sequences with ClustalW. Sequence 1 Product size (bp) Sequence 2 Product size (bp) Score AF PI AF Altay AF Aytın AF ES AF Harmankaya AF zgi AF Sönmez PI Altay PI Aytın PI ES PI Harmankaya PI zgi PI Sönmez Altay Aytın Altay ES Altay Harmankaya Altay zgi Altay Sönmez Aytın ES Aytın Harmankaya Aytın zgi Aytın Sönmez ES Harmankaya ES zgi ES Sönmez Harmankaya zgi Harmankaya Sönmez zgi Sönmez

4 Temel et al Table 3. Comparison of the second exon sequences with ClustalW. Sequence 1 Product size (bp) Sequence 2 Product size (bp) Score AF PI AF Altay AF Aytın AF ES PI Altay PI Aytın PI ES Altay Aytın Altay ES Aytın ES Figure 2. Phylogram tree of the first exon sequences. Figure 3. Phylogram tree of the second exon sequences. more conserved than the second one. Sequence variations could effect gene expression and could weaken yellow rust resistance. Research on yellow rust resistance genes performed by other authors (Sun et al., 2002; Chen et al., 2003; Yan et al., 2003; Yildirim et al., 2004; Li et al., 2006) generally depend on marker development. Wang et al. (2002) found out microsatellite markers such as Xpsp3000 linked to Yr10. These authors crossed PI with Yumai 18, a susceptible com-mon wheat variety from China and investigated inheritance of the Yr10 gene. They showed that the resistance to strain CYR31 was determined by a single dominant gene. Bozkurt et al. (2007) isolated RGAs using homology based PCR to target conserved regions (NBS) from bread wheat varieties. They found one RGA similar to Yr10 of wheat. A Yr10-like protein (RGAYr10) gene (GenBank No: EU428764) was identified in Dasypyrum breviaristatum recently (Tang and Yang, unpublished, NCBI). However, there is no report on Yr10 sequence polymorphisms in different wheat varieties. Although the PCR and sequencing results are not directly related to phenotypic data and cannot discern resistant/susceptible varieties, it might be striking that the least similar varieties Harmankaya99, zgi01 and Sönmez2001 lack the downstream region of the second exon. Detailed expression analyses would be helpful for the determination of most important nucleotide changes whether there is a relation with the constitutive expression of the Yr10 gene in these plants. We are planning to test these molecular data in F 2 generation to find out the relations with resistant genotypes selected in the field. It is thought that the results of this work will contribute to determine the divergence bet-

5 2332 Afr. J. Biotechnol. ween resistant and susceptible varieties and will be helpful to breeding applications. ACKNOWLEDGEMENT This work was supported by the Research Foundation of the Istanbul University; Projects No. T-840/ , UDP-/624/ and TUBITAK/TARAL 1007 Grant No. 105G075. REFERENCES Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990). Basic local alignment search tool. J Mol. Biol. 215(3): Bozkurt O, Hakki EE. Akkaya MS (2007). Isolation and sequence analysis of wheat NBS-LRR type disease resistance gene analogs using degenerate PCR primers. Biochem. Genet. 45(5/6): Chen X, Marcelo AS, Guiping Y, Jun S, Dubcovsky J (2003). Development of Sequence Tagged Site and Cleaved Amplified Polymorphic Sequence Markers for Wheat Stripe Rust Resistance Gene Yr5. Crop Sci. 43: Li GQ, Li ZF, Yang WY, Zhang Y, He ZH, Xu SC, Singh RP, Qu YY, Xia XC (2006). Molecular mapping of stripe rust resistance gene YrCH42 in Chinese wheat cultivar Chuanmai 42 and its allelism with Yr24 and Yr26. Theor. Appl. Genet. 112(8): Line RF, Chen XM (1995). Successes in breeding for and managing durable resistance to wheat rusts. Plant Dis. 79: Mallard S, Gaudet D, Aldeia A, Abelard C, Besnard AL, Sourdille P, Dedryver F (2005). Genetic analysis of durable resistance to yellow rust in bread wheat. Theor. Appl. Genet. 110(8): Peng JH, Fahima T, Roder MS, Huang QY, Dahan A, Li YC, Grama A, Nevo E (2000). High-density molecular map of chromosome region harboring stripe-rust resistance genes YrH52 and Yr15 derived from wild emmer wheat, Triticum dicoccoides. Genetica (The Hague) 109: Röbbelen G, Sharp EL (1978). Mode of inheritance, interaction and application of genes conditioning resistance to yellow rust. Fortschr. Pflanzenzücht. 9: Song W, Henry RJ (1995). Molecular analysis of the DNA polymorphism of wild barley (Hordeum spontaneum) germplasm using the polymerase chain reaction. Genet. Res. Crop Evol. 42(3): Spielmeyer W, Lagudah E (2003). Homoeologous set of NBS-LRR genes located at leaf and stripe rust resistance loci on the short arms of chromosome 1 of wheat. Funct Integr Genom. 3: Sun Q, Wei Y, Ni C, Xie, Yang T (2002). Microsatellite marker for yellow rust resistance gene Yr5 introgressed from spelt wheat. Plant Breed. 121: Tang Z, Yang Z (2008). Isolation and molecular diagnosis of a fulllength resistance gene analogue from Dasypyrum breviaristatum Thompson JD, Higgins DG, Gibson TJ (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22: Wang LF, Ma JX, Zhou RH, Wang XM, Jia JZ (2002). Molecular tagging of the yellow rust resistance gene Yr10 in common wheat, P.I (Triticum aestivum L.). Euphytica 124: Yan GP, Chen XM, Line RF, Wellings CR (2003). Resistance gene analog polymorphism markers co-segregating with the Yr5 gene for resistance to wheat stripe rust. Theor. Appl. Genet. 106: Yildirim A, Karadag Y, Sakin MA, Gokmen S, Kandemir N, Akkaya MS, Yildirim F (2004). Transfer of stripe rust resistance gene Yr26 to Turkish wheats using microsatellite markers. Cereal Res. Comm. 32(1):